The present invention is related to the suppression of non-biological motion of a cell. More specifically, the present invention is related to the suppression of non-biological motion of a cell having a viscosity enhancement medium, such as methyl cellulose.
Cell motility plays an important role in numerous cellular biological processes, for example immune response and modulation, stem cell engraftment in bone marrow transplantation, wound healing, biomaterials compatibility, tissue engineering, tumor metastasis, myocardial angiogenesis and tumor anti-angiogenesis, to name some areas of commercial interest with relevance for improving human health. In all of these cases, the measurement of cell motility in vitro provides a basis for better understanding the biology of the process and for testing the effects of pharmaceuticals or other therapeutic approaches with potential for assisting or inhibiting the process of cell motility.
Time-lapse imaging provides the most direct and informative method for analysis of motility in vitro, particularly for adherent cell types. Velocity and changes in velocity over time, direction of motion, persistence (tendency for motion in one direction), frequency of directional changes, frequency of stopping and starting, time spent in motion and at rest, total distance traveledxe2x80x94these are some of the parameters accessible to the automated time-lapse method of analysis that are not accessible by other means. The capability for dissecting out such features of motion is important for determining mechanisms of interaction of potentially therapeutic compounds, because different aspects of motion can be affected, depending upon the molecular pathway(s) involved (Ware, Wells, and Lauffenburger, 1998).
With time-lapse video, short-lived effects or transient effects of added compounds on motility can be readily quantified through comparison to baseline values up to the moment of compound addition. In cases of chemotactic behavior, the response may arise through signaling of xe2x80x9cdifferentialxe2x80x9d receptors, i.e. receptors that transmit intracellular signals only when ligand concentration changes (Dunn, 1990). In such cases, the response of the individual cell may depend upon the recent prehistory of the cell; time-lapse analysis reveals such behavior patterns.
Of particular interest to us is the possibility of screening for very short-lived secreted products on the basis of changes in migration patterns or morphology or phenotypic marker expression of cells in the immediate vicinity of transfected or otherwise engineered xe2x80x9csecretor cells.xe2x80x9d Such short-lived products may exist and have important roles in physiological processes, but being short-lived, they would not be readily detectible due to their instability under normal circumstances. Genes for such products could be transfected into xe2x80x9csecretor cellsxe2x80x9d that would express and secrete these products continuously in culture. Changes in motility or other phenotypic indicators of nearby cells would reveal the activity of such compounds. Examples of such compounds might include chemotactic agents, i.e. compounds that induce directed migration in cells. Such compounds guide cells to sites of relevant physiological interactions, for example in coordinating interaction of T cells, dendritic cells, and B cells in peripheral lymphoid tissues for immune response and in guiding neuronal cell synaptic connections during development of the nervous system.
In addition to the analysis of chemotactic responses to short-lived products, as described above, where in situ secretion from living cells would be necessary, we are also interested in analysis of chemotactic responses to more stable compounds, such as chemokines. Such compounds would be released in the vicinity of responding cells by non-biological methods, for example by impregnating gelatin beads or small microvessels or by application of chemotactic compounds to the culture surface.
In some cases, the migratory response to extracellular signaling molecules is linked to changes in cell adhesion molecules and in cell surface markers (phenotype). Moreover, it would be desirable to determine whether specific subpopulations of cells of similar phenotype show similar specific responses in motile behavior toward various stimuli. Linking surface marker phenotype analysis with motile behavior can feasibly be accomplished in parallel with imaging and intelligent image analysis. These goals hold tremendous potential as tools for investigative biology as well as screening of potentially therapeutic compounds.
The present invention addresses the development of capabilities for automated video time-lapse analysis of biological motility of adherent or non-adherent cells of all types. T lymphocytes from peripheral blood are used as a model system here. The category xe2x80x9cnon-adherentxe2x80x9d pertains broadly to cells of the hematopoietic system, including both lymphoid and myeloid lineages. Non-adherent cells can also exist in non-hematopoietic systems, such as freshly isolated myoblasts and certain cell lines (e.g. adapted HeLa (cervical adenocarcinoma) and PC-3 (prostate adenocarcinoma) cells, Colo 205 (colorectal adenocarcinoma), KNRK (normal kidney), RF-1 (gastric adenocarcinoma), Colo 587 (pancreatic adenocarcinoma), and others). Although some hematopoietic cells, most notably monocyte/macrophages and dendritic cells, adhere to tissue culture plastic, most hematopoietic cell types exhibit weak or transient attachment dependent upon added factors, e.g. phytohemaglutinin (PHA), serum components, fibronectin, or immobilized antibodies such as anti-CD3 for T lymphocytes.
Although the non-adherence of blood cells in vivo is implicit, the non-adherent nature in vitro of many types of hematopoietic cells is not so readily accepted. Some investigators hold that T-cells, for example, develop an adherent phenotype upon in vitro activation (consultant, personal communications). Most theories of cell migration and motility require the involvement of molecular attachment of cell adhesion molecules to the surface, for example through integrin-mediated binding to fibronectin (DiMilla et al., 1993; Lauffenburger, 1996; Maheshwari et al., 1999), and there is as yet no satisfactory theory for how non-adherent cells migrate. Nevertheless, observations over the course of numerous experiments, including round-the-clock imaging of CD34+lin-cord blood cells and their progeny, and experiments with naxc3xafve and prestimulated peripheral blood T lymphocytes, indicate that hematopoietic cells are highly animated and highly motile. However, it has also become clear that major components of the migratory xe2x80x9cbehaviorxe2x80x9d of these cells are non-biological influences of gravity and micro-turbulence, probably due to thermal convection. Convincing evidence for non-biological motion includes observation of dead (propidium iodide positive) cells moving separately in parallel with live cells. Similarly, the movement of beads and particles, the xe2x80x9cflockingxe2x80x9d or xe2x80x9cherdingxe2x80x9d of live cells for no apparent cause, and finally xe2x80x9cforward and reversexe2x80x9d tilting of the entire microscope by less than 3xc2x0 leave no question that the biological adherence of these cells is relatively weak in comparison to ambient factors such as gravity and turbulence. Yet, as described below, when these ambient factors are controlled, adherence-independent biological motion is clearly evident, and this motion is sensitive to the influence of relevant biological compounds.
Some examples presented in the literature of time-lapse video analysis of hematopoietic cell migration patterns represent, instead, typical examples of environmentally induced xe2x80x9cambient motilityxe2x80x9d (Crisa et al., 1996; Francis et al., 1997). The cited patterns are similar to those observed repeatedly in a variety of culture vessels with different types of hematopoietic cells, including, even, dead ones. In one of these reported studies, video time-lapse images were used to support an observed arrest of T-cell migration with anti-VLA4 or anti-VLA5 specific antibodies (Crisa et al., 1996). However, the xe2x80x9carrestxe2x80x9d of migration observed after antibody addition was timed in each case with the cessation of an initial wave of unidirectional motion lasting for 2.5 hours. In other words, the xe2x80x9carrestxe2x80x9d may have occurred without added antibody due to transient and variable ambient motion. In numerous experiments, such ambient motion has been observed as cells initially settle downhill into lower areas of the well. Motion stops when the majority of cells have passed beyond the viewfield. Given such problems, and despite the appeal of video time-lapse imaging for gathering otherwise unobtainable information relating to detailed characteristics of cell migration, there are as yet no validated methods described in the literature for 2 dimensional migration analysis of non-adherent cells.
A method for video time-lapse three-dimensional (3D) analysis of T cell migration has been reported (Friedl, Noble, and Zanker, 1995). This method is based upon the use of 3D collagen gels and does not allow for analysis of motion that is achieved apart from surface adhesion. These authors distinguish 3D from 2D analysis and state, xe2x80x9cOnto two-dimensional surfaces coated with ECM components, nonactivated peripheral T cells do barely adhere and are therefore incapable of migration. However, the incorporation of these cells into a 3D collagen environment leads to the onset of spontaneous migration; this results in the rapid and persistent tyrosine phosphorylation of FAK, implicating FAK in T cell migration.xe2x80x9d (Entschladen et al., 1997). This quote confirms the generally held assumption that without adherence, there is no migration. No explanation is offered as to why T cells do not adhere to ECM (extracellular matrix) components coated onto a 2D surface, i.e. tissue culture plastic, and yet T cells do apparently adhere when incorporated into a 3D collagen matrix.
It is suggested that the failure to adequately control ambient motion is the reason why a validated, reproducible method has not yet been put forth for analysis of motion in a 2D environment with non-adherent cells. In the presence of a very slight tilt (less that 30), motion is observed to trend downward, and if slight convection is present, live cells are observed to follow the direction of flow of particles and dead cells. This ambient motion is superimposed upon their active motility. Perhaps upon observing this, other researchers recognize first that there is no strong adherence, and then it may be assumed that all xe2x80x9cresidualxe2x80x9d motion is thermal or biologically irrelevant. However, as described more fully below, clearly relevant biological motion is seen using methyl cellulose in 2D cultures.
Likewise, no method has been presented for analysis of 3D motion in the absence of a solid matrix (e.g. collagen) upon which cells can attach. However, 3D motion among T cells in the absence of a solid support has been observed using methyl cellulose at a concentration of 1.2% (see below). Also, 3D motion in video time-lapse images of myeloid cell subpopulations in typical CFU-GM cultures has been observed using methyl cellulose at a concentration of approximately 0.9%.
Methyl cellulose has been widely applied for the purpose of growing xe2x80x9ccoloniesxe2x80x9d of cells. Colonies are small clumps or groups of cells; they are presumed to arise from a single cell, and are used as a measure of xe2x80x9ccolony forming unitsxe2x80x9d (CFU). The ability of a cell to form a colony is equated with its being a xe2x80x9cprogenitorxe2x80x9d cell, and so CFU type assays are also known as progenitor cell assays. The methyl cellulose allows the formation of a colony to proceed over a one to two-week period in culture without mixing or disruption of the cell positions.
In summary, there are apparently no validated methods in the literature for analysis of migration of T cells or other non-adherent cells on a 2D surface or for analysis of migration in 3 dimensions when there is no solid matrix on which the cells can attach. When methyl cellulose is used, this dissolved compound is not considered to provide attachment surfaces for the cells to adhere to. There may be molecular attachment involved, but there is no apparent requirement for it, because the cells move in medium alone without methyl cellulose where, in the absence of strong ambient motion, they can be observed to xe2x80x9cswimxe2x80x9d just the same as in methyl cellulose-containing solution. In medium alone, frequently it is difficult to distinguish biological from thermal and other types of ambient motion, and in many cases the ambient motion is not of uniform direction across the viewfield, nor is it constant over time. Therefore, methods to mathematically xe2x80x9ccorrectxe2x80x9d for ambient motion will have noise (uncertainty in precision) associated with them, and in many cases this noise will be greater than the magnitude of biological motion. With methyl cellulose, a physical method for suppression of ambient non-biological motion on a 2D surface has been developed when there is no attachment involved.
Interestingly, as reviewed by Wilkinson (Wilkinson, 1990), prior to development of the filter assay (see below), xe2x80x9cmany beautiful studiesxe2x80x9d of leukocyte motion were made using video photography and these studies xe2x80x9claid the foundations on which contemporary studies are based.xe2x80x9d But due to the degree of technical difficulty, these visual methods were abandoned when the Boyden filter assay, now commonly known as the xe2x80x9cBoyden chamberxe2x80x9d (Boyden, 1962; Falk, Goodwin, and Leonard, 1980) or xe2x80x9cTranswell migration assayxe2x80x9d (Bleul et al., 1996), was introduced. The vast majority of current motility and chemotaxis investigation is conducted using this method, whereby cells are added to a chamber separated by a membrane from a second chamber containing medium with test compounds. The cells migrate through small well-defined pores into the lower chamber, and after a specified interval, they are counted and compared with background numbers of cells migrating into chambers without added compounds. While an abundance of valuable data has been obtained using the Boyden chamber, this method xe2x80x9calso had the drawback that it was now possible to spend a research career studying leukocyte chemotaxis without ever looking at a moving cell or, indeed, knowing its front from its back xc2xcxe2x80x9d, according to Wilkinson (Wilkinson, 1990). Also with introduction of the filter method, many of the clear distinctions regarding chemotaxis, chemokinesis, contact guidance, direction reactions, and other forms of locomoter reactions became blurred. Nevertheless, the filter assay is seeing tremendous application in the discovery of a large family of chemotactic compounds known as xe2x80x9cchemokinesxe2x80x9d (Allavena et al., 1999; Hedrick and Zlotnik, 1999), which hold interest for therapeutic application both in terms of the ligands themselves and in terms of their receptors as targets for small drug molecules. The answers to questions about the exact role of each of these chemokines in the host immune response will be better answered with analytical approaches such as described by the present invention.
In the 1970""s, the xe2x80x9cunder-agarose assayxe2x80x9d was introduced. An agarose layer was poured over a glass slide to form a gel, then holes were carefully bored and the agarose plugs were removed down to the surface of the glass slide (Nelson, Quie, and Simmons, 1975). Cells were introduced into one hole and chemotactic compounds or control substances could be introduced into the other holes. Over time, the cells were seen to migrate between the agarose and the glass toward a chemotactic compound at a faster rate than toward a neutral control substance. Whereas the Boyden filter assay yields only relative cell numbers, corresponding to relative chemotactic strength, the xe2x80x9cunder-agarose assayxe2x80x9d yielded a distance traveled over time for the cell migration front. This distance was originally compared to the distance traveled by the control front on the other side of the well toward the neutral compound to derive an index of migration. This simplistic approach to migration analysis stimulated valuable mathematical treatment of the problem (Farrell, Daniele, and Lauffenburger, 1990; Lauffenburger, Rothman, and Zigmond, 1983; Nagahata et al., 1991; Rothman and Lauffenburger, 1983; Rupnick et al., 1988; Stickle, Lauffenburger, and Zigmond, 1984), which has provided the mathematical framework for much current thinking in this area. However, the method is subject to variability depending upon how the holes are bored, perhaps due to lifting of the agarose from the glass surface allowing cells to migrate along with channeling fluid rather than through biological motility.
Another method in current use is measurement of the infiltration of lymphocytes into a 3 dimensional collagen gel (Friedl, Noble, and Zanker, 1995; Nikolai et al., 1998; Nikolai G, 1995). Cells are cultured in contact with the gel surface, and after an elapsed time period, cells migrating to a certain depth are counted. This number correlates to cytokinetic activity.
Although all of these methods provide a quantitative measure of migratory activity, their shortcomings leave many aspects of the migratory behavior hidden from the investigator.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution having a viscosity enhancement medium. There is the step of measuring the motility of the cell. Multiple cells can be measured in parallel.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of measuring the motility of the cell in the solution when there is no attachment of the cell involved.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps placing the cell in a solution. There is the step of identifying and quantifying short lived effects or transient effects of added moiety on motility of the cell in the solution.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution having a viscosity of about 100-5000 centipose. There is the step of screening for short-lived secreted products from the cell as a function of changes in migration patterns or morphology or phenotypic marker expression of the cell adjacent to transected or otherwise engineered secretor cells or the cells themselves.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution having a viscosity of about 100-5000 centipose. There is the step of linking surface marker phenotype analysis of the cell in the solution with motile behavior of the cell in the solution.
The present invention pertains to a method for analyzing cells. The method comprises the steps of placing the cells in a solution having a viscosity of about 100-5000 centipose. There is the step of linking surface marker phenotype analysis of adherent and non-adherent cells in the solution with motile behavior of the adherent and non-adherent cells in the solution.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution having a viscosity of about 100-5000 centipose. There is the step of performing two-dimensional or three-dimensional migration analysis on the cell in the solution.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution having a viscosity of about 100-5000 centipose. There is the step of analyzing migration of the cell in the solution which occurs without adherence.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of controlling ambient motion of the cell in the solution as a reproducible method for analysis of motion in a 2D or 3D environment with non-adherent cells.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of analyzing 3D motion of the cell in the solution in the absence of a solid matrix upon which the cell can attach.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of suppressing the ambient non-biological motion of the cell in the solution on a 2D surface when there is no attachment involved of the cell.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution having a viscosity of about 100-5000. There is the step of measuring motility of the cell in the solution, where surface attachment by the cell is not utilized.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of forming a thin film in the solution whose viscosity resists Brownian and other non-biological sources of motion but does not interfere with active cell biological motion.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of adding a protein or other biological or chemical moiety to the solution. There is the step of analyzing the effect of the protein on cell motility, morphology, phenotype, division rate, cell death, or blebbing or disease state.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps placing the cell in a solution. There is the step of adding a protein to the solution. There is the step of analyzing the protein function regarding the cell using cell motility as an analytical marker.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of placing methyl cellulose in the solution to reduce ambient motion of the cell in the solution and eliminate convective motion.
The present invention pertains to a method for suppressing non-biological movement of a cell. The method comprises the steps of placing the cell in a solution. There is the step of forming a layer of methyl cellulose 34 to 137 Um thick in the solution.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of using methyl cellulose in the solution for stopping the effects of gravity on the cell in the solution.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of using methyl cellulose in the solution for reducing or eliminating the effects of micro-turbulances due to thermal convection in the solution.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of introducing methyl cellulose in the solution for stopping motion of the cells due to mechanical movement of a plate on which the cells are disposed.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of introducing a viscous fluid having a viscosity of about 100-5000 centipose in the solution for stopping or reducing the effects of gravity on the cell.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of introducing a viscous fluid having a viscosity of about 100-5000 centipose in the solution for reducing the effects of micro-turbulences due to thermal convection.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of introducing a viscous fluid having a viscosity of about 100-5000 centipose in the solution for stopping motion of the cells due to mechanical movement of the plate.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps of placing the cell in a solution. There is the step of using methyl cellulose or any viscous fluid to separate biological motility from ambient motility.
The present invention pertains to a method for analyzing a cell by suppressing non-biological movement. The method comprises the steps placing the cell in a solution. There is the step of measuring biological cell motility with the cell in the solution.
The present invention pertains to a method for analyzing cells. The method comprises the steps of placing the cells in a solution. There is the step of measuring biological cell motility for adherent or nonadherent cells in the solution.
The present invention pertains to a method for analyzing cells. The method comprises the steps of placing the cells in a solution. There is the step of measuring biological motility of both adherent and nonadherent cells using visible and fluorescent images.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of linking a surface marker of the cell in the solution by phenotype analysis with motile behavior. The linking step an include the step of linking a surface marker of the cell in the solution by phenotype with motile behavior.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of measuring swimming vs moving of cells in the solution in a 2D plane, as cells move up into a viscous layer of the solution.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of measuring attachment of the cell to a surface in the solution, by dispensing fluid into the solution and looking for a location where the cell detaches from the surface.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of measuring the effect tilt has on cell motion, by changing the angle a plate is tilted on which the cell is disposed and looking for changes in motion or cell attachment of the cell.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of removing methyl cellulose effects in the solution by mixing the solution and diluting the methyl cellulose.
The present invention pertains to a method for analyzing a cell. The method comprises the steps placing the cell in a solution having methyl cellulose. There is the step of removing the methyl cellulose from the solution. There is the step of treating the cell with a desired material. There is the step of reintroducing the methyl cellulose into the solution.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution having a viscosity of between 100-5000 centipose. There is the step of measuring cell division, morphology, cell phenotype, disease state of the cell, or cell death. There can also be the step of measuring the motility of the cell.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of holding a cell intact for suppressing motility for division detection of the cell in the solution.
The present invention pertains to a method for analyzing a cell. The method comprises the steps of placing the cell in a solution. There is the step of analyzing migratory response of the cell to extracellular signaling molecules linked to changes in cell adhesion molecules and in cell surface markers (phenotype).
The present invention pertains to a method for analyzing cells. The method comprises the steps of placing the cells in a solution. There is the step of identifying specific subpopulations of cells of similar phenotype which show similar specific responses in motile behavior toward various stimuli.
The present invention pertains to a method for analyzing cells. The method comprises the steps of placing the cells in a solution. There is the step of identifying and separating specific subpopulations of the cells based on cell phenotype, morphology, motility, cell proliferation, cell death, or disease state.